Many pathogens rapidly mutate and recombine, making it difficult to produce effective vaccines. This problem is most prevalent with viral pathogens, and in particular with RNA viruses, such as HIV. HIV infected individuals can harbor multiple HIV strains and typically harbor multiple HIV quasispecies. Therapeutic strategies aimed at treating individuals infected with pathogens such as HIV-1 face enormous difficulties ranging from the development of viral strains which are resistant to the anti-virals to the numerous side effects of these drugs, making continuous administration of these therapies extremely difficult and expensive.
Several therapeutic HIV vaccines have been developed, however clinical trials of these vaccines have shown little efficacy. One explanation for the lack of success of these vaccines is the use of viral vectors as immunogens which encompass consensus sequences of the HIV viral genome. The presence of consensus sequences does not necessarily argue for the efficacy of the sequences in eliciting immune responses against a heterologous virus. Because of the still high and growing level of divergence between the consensus sequence and the autologous infecting HIV strain, vaccines or anti-viral reagents based on the HIV-1 subtype B consensus sequence are unable to adequately treat HIV infection. Moreover, it is likely that those viral vector-based vaccines do not target the most efficient cells of the immune system in terms of antigen processing and presentation, namely dendritic cells.
A number of publications have suggested the use of antigen presenting cells loaded with HIV nucleic acids or peptides as a vaccine for HIV. For example, Huang et al. (J. Infect. Dis. 2003 187:315-319) disclose dendritic cells loaded with liposome-complexed HIV proteins and the possible use of such loaded cells as a vaccine. Weissman et al. (J Immunol 2000 165:4710-4717) disclose transfection of dendritic cells with mRNA encoding a single cloned HIV gag sequence, resulting in the delivery of antigenic gag peptides to MHC class I and II molecules and the induction of CD4+ and CD8+ T cells responses in vitro. U.S. Pat. No. 5,853,719 and U.S. Pat. No. 6,670,186 (Nair et al.) describe the use of RNA derived from a tumor or pathogen isolated from a patient for in vitro loading of autologous antigen presenting cells, and their use as a vaccine. However, vaccinating patients with antigen presenting cells loaded with total pathogen RNA could increase the pathogen load in the patient.
The difficulty in selecting primers capable of amplifying the nucleic acids from all variants of a given pathogen, and in particular, all variants of HIV, has long been recognized. Amplification of specific regions of the HIV genome is complicated by the high mutation rate of the HIV genome caused by the low fidelity reverse transcriptase of the virus and immune selection in vivo. Nevertheless, primers have been developed that can amplify certain regions of the HIV genome from multiple HIV variants. For example, Abravaya et al. (J Clinical Microbio 2000 38:716-723) disclose HIV-1-specific and HIV-2-specific primers and probes targeted to conserved sequences in the pol gene for use in multiplex PCR assays to detect HIV-1 group M subtypes A, B, C, D, E, F and G, and group O and HIV-2 RNA in plasma, and the use of such assays to improve the safety of the blood supply.
Christopherson et al. (Nucleic Acids Res 1997 25:654-658) discuss the effect of internal primer-HIV-1 template mismatches on RT-PCR to amplify a 142 base pair region of gag. Five to six mismatches, but not two to four mismatches, between the HIV-1 template and primers 28-30 bases length significantly decreased the yield of RT-PCR. The primers were designed to be longer than is typically used in order to accommodate mismatches. Reduced efficiency of amplification of the more divergent HIV-1 subtypes A and E could be improved 4-fold to 10-fold by lowering the annealing temperature to 50° C. and implementing a reverse transcription step that gradually increases in temperature. Also, substitution of 5-methylcytosine for cytosine, or of inosine at positions of variable bases resulted in less than 4-fold difference in product yield between the homologous and most divergent HIV-1 templates. Primers that terminated in a T allowed amplification when matched or mismatched with C, G or T.
Michael et al. (J Clin Microbiol 1999 37:2557-2563) disclose primers for the amplification of a 155 nucleotide sequence of the HIV-1 gag gene. The primers were selected to maximize homology to 30 HIV-1 isolates of subtypes A through G, and to minimize HIV-1 to primer mismatches near the 3′ end of the primer. The annealing temperature during amplification was lowered to increase mismatch tolerance. The optimized primers and amplification conditions increased the quantity of HIV-1 RNA detected in comparison to previously available tests, but underrepresented subtypes A, D, F and G, and contained no members of subtypes H and J.
U.S. Pat. No. 6,001,558 discloses the use of multiple primers to amplify short fragments (<200 base pairs) of the LTR and pol regions from multiple HIV-1 isolates and the LTR, pol and env regions from multiple HIV-2 isolates. Multiple probes were then used to detect the amplification products. The primer/probe combinations were able to detect five HIV-1 isolates from groups M and O, and two HIV-2 isolates. Primers corresponding to highly conserved sequences of HIV-1 were selected by the following criteria: 1) the lengths of amplified PCR products could not exceed 200 base pairs, as smaller products are more efficiently amplified than larger products, and are less sensitive to other reaction conditions; 2) primer sets must amplify a functional probe region for detection of the amplified product; 3) mismatch near the 3′ end of the primer would be far less preferable than mismatches near the 5′ end; and 4) other criteria for 3′ end stability, length (about 23-31 nucleotides), GC content and interactions with other primers and probes.
U.S. Pat. No. 6,194,142 discloses methods for in vitro diagnosis HIV-1, HIV-2 and SIV infection using primer pairs corresponding to sequences that are conserved between the gag, pol and env genes of HIV-1 Bru, HIV-1 Mal and HIV-1 Eli, HIV-2 ROD and SIV MAC strains, and between the nef2, vif2 and vpx genes of HIV-2 ROD and SIV MAC, or the env, nef1, vif1 and vpr genes of HIV-1 Bru, HIV-1 Mal and HIV-1 Eli. Mixtures of primers with variant nucleotides corresponding to HIV variants and SIV were used simultaneously in a PCR reaction.
U.S. Pat. No. 6,232,455 discloses primer/probe sets derived from consensus sequences of 31 HIV-1 group M (subtypes (A, B and D) and group O isolates and 14 isolates of the HIV-2 A and B subtypes. HIV-1 consensus primer/probe sets are specific for the detection of HIV-1, while HIV-2 consensus primer/probe sets are specific for the detection of HIV-2.
U.S. Pat. No. 6,531,588 discloses primers for amplification of 0.7 kB, 1.57 kb or 2.1 kb regions of pol from multiple HIV quasispecies in a patent, followed by sequencing to determine patient-specific HIV genotype information for use in determining the appropriate therapy and monitoring drug resistance.
U.S. 2003/0148280 disclose primer sets and probes for the detection of purportedly all HIV-1 group M, N and O strains including circulating recombinant forms (CRF, which refers to recombinants between subtypes), and inter-group recombinants. The primers are targeted to HIV-1 gag p24 (399 bp amplicon), pol integrase (864 bp amplicon), and env gp41 immunodominant region (IDR; 369 bp amplicon).
None of these publications suggest the possibility of using amplified autologous pathogen nucleic acids encoding specific polypeptides from multiple species of a pathogen present in an individual for the preparation of antigen presenting cell vaccines or nucleic acid vaccines. The present invention addresses the long felt need to develop patient specific vaccines to treat autologous infecting pathogens and offers additional advantages as well.